This invention relates to the preparation and use of nucleic acid fragments or genes which encode fungal palmitoyl-CoA xcex94-9 desaturase enzymes to create transgenic plants having altered oil profiles.
Plant-produced oils can be found in a wide variety of products including lubricants and foods. Interestingly, different plant species synthesize various oil types. For example, coconut and palm plants produce oils that are abundant in fatty acids having medium chain lengths (10-12 carbon atoms). These oils are used in manufacturing soaps, detergents and surfactants, and represent a U.S. market size greater than $350 million per year. Other plants, such as rape, produce oils abundant in long chain fatty acids (22 carbon atoms) and are used as lubricants and anti-slip agents. Additional applications of plant oils include their use in plasticizers, coatings, paints, varnishes and cosmetics (Volker et al., (1992) Science 257:72-74; Ohlrogge, (1994) Plant Physiol. 104:821-826). However, the predominant use of plant oils is in the production of food and food products.
Over the years, vegetable-derived oils have gradually replaced animal-derived oils and fats as the major source of dietary fat intake. However, saturated fat intake in most industrialized nations has remained at about 15% to 20% of total caloric consumption. In efforts to promote healthier lifestyles, the United States Department of Agriculture(USDA) has recently recommended that saturated fats make up less than 10% of daily caloric intake. To facilitate consumer awareness, current labeling guidelines issued by the USDA now require total saturated fatty acid levels be less than 1.0 g per 14 g serving to receive the xe2x80x9clow-satxe2x80x9d label and less than 0.5 g per 14 g serving to receive the xe2x80x9cno-satxe2x80x9d label. This means that the saturated fatty acid content of plant oils needs to be less than 7% and 1.75% to receive the xe2x80x9clow satxe2x80x9d and xe2x80x9cno satxe2x80x9d label, respectively. Since issuance of these guidelines, there has been a surge in consumer demand for xe2x80x9clow-satxe2x80x9d oils. To date, this has been met principally with canola oil, and to a much lesser degree with sunflower and safflower oils.
The characteristics of oils, whether of plant or animal origin, are determined predominately by the number of carbon and hydrogen atoms, as well as the number and position of double bonds comprising the fatty acid chain. Most oils derived from plants are composed of varying amounts of palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2) and linolenic (18:3) fatty acids. Conventionally, palmitic and stearic acids are designated as xe2x80x9csaturatedxe2x80x9d since the fatty acid chains have 16 and 18 carbon atoms, respectively, and no double bonds. They therefore contain the maximal number of hydrogen atoms possible. However, oleic, linoleic, and linolenic are 18-carbon fatty acid chains having one, two, and three double bonds, respectively, therein. Oleic acid is typically considered a mono-unsaturated fatty acid, whereas linoleic and linolenic are considered to be poly-unsaturated fatty acids.
Saturated fatty acids are linear molecules and tend to form self-stacked structures thereby resulting in high melting temperatures; a characteristic that is quite desirable when producing foods like chocolate. Animal fats, which are also solid at room temperature, are another readily available source of saturated fatty acids. However, use of said oil is often discouraged due to the high levels of cholesterol associated therewith. In comparison, unsaturated fatty acid chains are nonlinear due to bending induced by double bond insertion. The bending of the molecule impedes the ability of the fatty acid chains to stack thus causing them to remain fluid at lower temperatures. Vegetable oils, for example, are high in unsaturated fatty acids, and therefore are typically liquid at room temperature. Furthermore, saturated fatty acid can be modified to become unsaturated fatty acids by removal of hydrogen atoms and insertion of double bonds between two carbon atoms on the fatty acid chain. Desaturation can be achieved either enzymatically or chemically and decreases melting points due to the inability of the fatty acid molecules to self-stack.
The total saturated fatty acid level of corn oil, averaging about 13.9%, does not meet the current labeling guidelines discussed above. On average, corn oil is comprised of 11.5% palmitic acid, 2.2% stearic acid, 26.6% oleic acid, 58.7% linoleic acid, and 0.8% linolenic acid. Corn oil also contains 0.2% arachidic acid, a twenty-carbon saturated fatty acid (Dunlap et. al., (1995) J. Amer. Oil Chem. Soc. 72:981-987). The composition of corn oil instills it with properties that are most desirable in edible oils. These include properties such as heat stability, flavor, and long shelf life. However, consumer demand for xe2x80x9clow satxe2x80x9d oils has resulted in a significant decrease in corn oil utilization and thus decreased market share. Therefore, a corn oil with modified levels of saturated fatty acids is highly desirable and would have practical use in that it would meet the consumer demand for healthier oils while having most or all of the properties that made corn oil a popular and preferred oil in the past.
Corn is typically not considered to be an oil crop as compared to soybean, canola, sunflower and the like. In fact, the oil produced and extracted from corn is considered to be a byproduct of the wet milling process used in starch extraction. Because of this, there has been little interest in modifying the saturate levels of corn oil until those efforts disclosed herein.
As disclosed herein, saturate levels of fatty acids comprising plant oils can be altered by expressing a fungal palmitate-CoA xcex94-9 desaturase within a plant cell. These proteins most likely enzymatically desaturate palmitate-CoA molecules by removing two hydrogen atoms and adding a double bond between the 9th and 10th carbon atoms from the CoA portion of the molecule, thus producing palmitoleic-CoA (16:1xcex949) . The palmitoleic-CoA is ultimately incorporated into seed oil thus lowering the total saturate levels of said oil.
In the present invention, a gene encoding a fungal palmitate-CoA xcex94-9 desaturase has been isolated and cloned from Aspergillus nidulans. The saturate level of oils found in plant cells can be altered by expressing said palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans. 
One aspect of the disclosed invention is a gene encoding said palmitate-CoA xcex94-9 desaturase, said gene being isolated and purified from Aspergillus nidulans. 
An additional aspect of the present invention relates to producing a gene wherein the codon bias of a gene from a non-plant source has been modified to look similar to genes from a plant source.
Another aspect of the invention relates to altering oil saturate levels within a plant cell by expressing said genes encoding palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans. Genes disclosed herein can be used to alter saturate levels by placing said genes in the sense orientation. Plants cells being transformed with genes encoding palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans in the sense orientation results in the oils of said plants having increased 16:1 levels and decreased total saturate levels over non-transformed plants.
An additional aspect of the present invention is the production of chimeric genes using the genes disclosed herein encoding for palmitoyl CoA-xcex94-9 desaturase in combination with promoter regulatory elements and the use of said chimeric genes within a plant cell.
Yet an additional aspect of the present invention is the transformation of plant species disclosed herein with said chimeric genes.
Other aspects, embodiments, advantages, and features of the present invention will become apparent from the following specification.
The present invention relates to methods and compositions for obtaining transgenic plants wherein plant oils produced thereby have altered saturate levels. The following phrases and terms are defined below:
By xe2x80x9caltered saturate levelsxe2x80x9d is meant that the level of total saturated fatty acids of a plant oil produced by a modified plant is different from that of a normal or non-modified plant.
By xe2x80x9ccDNAxe2x80x9d is meant DNA that is complementary to and derived from a mRNA.
By xe2x80x9cchimeric DNA constructionxe2x80x9d is meant a recombinant DNA containing genes or portions thereof from one or more species.
By xe2x80x9ccomplementarityxe2x80x9d is meant a nucleic acid that can form hydrogen bond(s) with other nucleic acid sequences either through traditional Watson-Crick or other non-traditional types of base paired interactions.
By xe2x80x9cconstitutive promoterxe2x80x9d is meant promoter elements that direct continuous gene expression in all cell types and at all times (i.e., actin, ubiquitin, CaMV 35S, 35T, and the like).
By xe2x80x9cdevelopmental specificxe2x80x9d promoter is meant promoter elements responsible for gene expression at specific plant developmental stages, such as in early or late embryogenesis and the like.
By xe2x80x9cenhancerxe2x80x9d is meant nucleotide sequence elements which can stimulate promoter activity such as those from maize streak virus (MSV) protein leader sequence, alfalfa mosaic virus protein leader sequence, alcohol dehydrogenase intron 1, and the like.
By xe2x80x9cexpressionxe2x80x9d as used herein, is meant the transcription and stable accumulation of mRNA inside a plant cell. Expression of genes also involves transcription of the gene to create mRNA and translation of the mRNA into precursor or mature proteins.
By xe2x80x9cforeignxe2x80x9d or xe2x80x9cheterologous genexe2x80x9d is meant a gene encoding a-protein whose exact amino acid sequence is not normally found in the host cell, but is introduced by standard gene transfer techniques.
By xe2x80x9cgenexe2x80x9d is meant to include all genetic material involved in protein expression including chimeric DNA constructions, genes, plant genes and portions thereof, and the like.
By xe2x80x9cgenomexe2x80x9d is meant genetic material contained in each cell of an organism and/or virus and the like.
By xe2x80x9cinducible promoterxe2x80x9d is meant promoter elements which are responsible for expression of genes in response to a specific signal such as: physical stimuli (heat shock genes); light (RUBP carboxylase); hormone (Em); metabolites, chemicals, stress and the like.
By xe2x80x9cmodified plantxe2x80x9d is meant a plant wherein the gene, mRNA, or protein from Aspergillus nidulans palmitate-CoA xcex94-9 desaturase is present.
By xe2x80x9cplantxe2x80x9d is meant a photosynthetic organism including both eukaryotes and prokaryotes.
By xe2x80x9cpromoter regulatory elementxe2x80x9d is meant nucleotide sequence elements within a nucleic fragment or gene which controls the expression of that nucleic acid fragment or gene. Promoter sequences provide the recognition for RNA polymerase and other transcriptional factors required for efficient transcription. Promoter regulatory elements from a variety of sources can be used efficiently in plant cells to express gene constructs. Promoter regulatory elements are also meant to include constitutive, tissue-specific, developmental-specific, inducible promoters and the like. Promoter regulatory elements may also include certain enhancer sequence elements and the like that improve transcriptional efficiency.
By xe2x80x9ctissue-specificxe2x80x9d promoter is meant promoter elements responsible for gene expression in specific cell or tissue types, such as the leaves or seeds (i.e., zein, oleosin, napin, ACP, globulin and the like).
By xe2x80x9ctransgenic plantxe2x80x9d is meant a plant expressing a chimeric gene introduced through transformation efforts.
In plant cells, fatty acids are made as acyl-acyl carrier protein (acyl-ACP) substrates and are elongated by various enzymes through the addition of malonyl-ACP to make acyl-ACP molecules ranging in length from 2 to 18 carbon atoms. Afterwards, acyl-ACP thioesterases catalyze the hydrolytic cleavage of palmitic acid, stearic acid, and oleic acid from ACP, in a somewhat selective although not specific manner, thus producing a free fatty acids. The fatty acid molecules move out of the plastid into the cytoplasm where they are eventually modified into acyl-CoA molecules. Said molecules are then incorporated onto the triglyceride oil fraction. It has been discovered by applicants as disclosed herein that desaturation of an acyl-CoA molecule, wherein said molecule is preferably stearoyl-CoA and most preferably palmitate-CoA, can reduce saturate levels in the triglyceride oil fraction. Said desaturation most preferably results in the production and accumulation of palmitoleic acid (16:1xcex94-9). Said desaturation may also result in a decrease in palmitic and stearic acid in the triglyceride oil fraction.
In corn seed oil, the predominant fatty acids are linoleic acid (18:2 at about 59%), oleic acid (18:1 at about 26%) and palmitic (16:0 at about 11%), with stearic acid (18:0) generally comprising about 2.5% or less (Glover and Mertz, (1987) in: Nutritional Quality of Cereal Grains: genetic and agronomic improvement., p.183-336, (eds. Olson, R. A. and Frey, K. J.) Amer. Soc. Agronomy, Inc., Madison, Wis.; Fitch-Haumann, (1985) J. Am. Oil. Chem. Soc. 62:1524-1531). Biosynthesis of fatty acids in plant cells is initiated in the plastids where they are synthesized as acyl-ACP thioesters by a fatty acid synthase complex. More specifically, fatty acid production is accomplished by a series of condensation reactions involving addition of malonyl-ACP sequentially to a growing fatty acid-ACP chain by the enzyme xcex2-ketoacyl-ACP synthase I (KAS I). Most fatty acid-ACP units reach carbon chain lengths of 16 and are then elongated to 18 carbon units by KAS II. The vast majority of C18 fatty acids become desaturated by stearoyl-ACP xcex94-9 desaturase at the C9 position from the carboxyl end to produce oleyl-ACP.
Both saturated and unsaturated fatty acid-ACP units are hydrolyzed by acyl-ACP thioesterases to produce free fatty acids. These free fatty acids then cross the plastid membrane to the cytosol of the cell where they are modified by addition of a CoA moiety. Afterwards, said fatty acids are incorporated into plant oils (Somerville and Browse, (1991) Science 252:80-87; Browse and Sommerville (1991) Annu. Rev. Plant Physiol. Plant Mol. Biol. 42:467-506; Harwood (1989) Critical Reviews in Plant Sci. 8:1-43; Chasan (1995) Plant Cell 7:235-237; Ohlrogge (1994) Plant Physiol. 104:821-826).
The palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans desaturates palmitic acid at the C9 position relative to the carboxyl end most likely after the point of modification with Co-A. In plant cells, this most likely occurs before being incorporated into the triglyceride fraction of the oil. Therefore, expressing palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans in plant-cells will cause a decrease in the saturate levels of the oil produced by said plant.
The palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans disclosed herein can be used to modify saturate levels in oil in both monocotyledonous and dicotyledonous plants. In dicotyledonous plants, expression of said desaturase preferably results in a decrease in 16:0 and 18:0 levels found in oil derived from said plants. More preferably, expression of said desaturase results in increased levels of 16:1 fatty acid in said oil. In monocotyledonous plants, expression of said desaturase preferably results in decreased levels of 18:0 and more preferably, increased levels of 16:1 found in the said oil. It is not applicants intention, however, to limit said gene expression exclusively to plants in that said desaturase and genes thereof can be expressed and used to modify lipid contents in both yeast and bacteria.
As further described herein, an Aspergillus palmitate-CoA xcex94-9 desaturase can be used to modify the saturate levels in oils produced by transgenic plants. Preferably, genes and nucleic fragments encoding the palmitate-CoA xcex94-9 desaturase are derived from Aspergillus nidulans. More preferably, genes encoding palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans are those disclosed herein as SEQ ID NO:5 and SEQ ID NO:12, said genes encoding a protein having an amino acid sequence as disclosed herein as SEQ ID NO:6.
One method by which plant oils can be modified is by expressing the palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans in a dicotyledonous plant. This can be achieved by placing the genes or nucleic acid fragments encoding said proteins in the sense orientation 3xe2x80x2 to a promoter regulatory element of choice followed by a transcriptional terminator at the 3xe2x80x2 end of said gene thus producing a chimeric gene construct. These chimeric genes can then be transformed into plants, thereby producing plant oils having altered saturate levels relative to nontransformed controls. Expressing the palmitate-CoA xcex94-9 desaturase as disclosed herein from Aspergillus nidulans in dicotyledonous plants results in plant oils derived therefrom having 16:1 levels as a percentage of the total fatty acid from about 0.23 to about 4.65%; preferably from about 3.01 to about 4.65%; more preferably from about 4.07 to about 4.65%, with about 4.65% being most preferred. The total saturate levels range preferably from about 9.8 to about 12.5% with about 9.8% being most preferred.
Another method by which plant oils can be modified is by expressing the palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans in a monocotyledonous plant. As with dicotyledonous plants, this can be achieved by placing the genes or nucleic acid fragments encoding said proteins in the sense orientation 3xe2x80x2 to a promoter regulatory element of choice followed by a transcriptional terminator at the 3xe2x80x2end of said gene thus producing a chimeric gene construct. These chimeric genes can then be transformed into plants, thereby producing plant oils having altered saturate levels relative to nontransformed controls. Expressing the palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans in monocotyledonous plants results in plant oils derived therefrom to have 16:1 levels from about 0.4 to about 3.2%; preferably from about 1.2 to about 3.2%, with about 3.2% being most preferred.
As further disclosed herein, chimeric gene constructs encoding palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans can be transformed in other oilseed crops to modify the saturate levels therein. Said oilseed crop plant species which may be modified include but are not limited to soybean, Brassicaceae sp., canola, rape, sunflower, flax, safflower, coconut, palm, olive, peanut, cotton, castor bean, coriander, Crambe sp., Cuphea sp., Euphorbia sp., Oenothera sp., jojoba, Lesquerella sp., marigold, Limnanthes sp., Vernonia sp., Sinapis alba, and cocoa, with maize being most preferred. Most if not all of these plant species have been previously transformed by those having ordinary skill in the art.
To obtain high expression of heterologous genes in plants it may be preferred to reengineer said genes so that they are more efficiently expressed in the cytoplasm of plant cells. Maize is one such plant where it may be preferred to reengineer the heterologous gene(s) prior to transformation to increase the expression level thereof in said plant. Therefore, an additional step in the design of genes encoding said palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans is the designed reengineering of a heterologous gene for optimal expression.
One reason for the reengineering the xcex94-9 Co-A desaturase gene from Aspergillus nidulans for expression in maize is due to the non-optimal G+C content of the native gene. For example, the very low G+C content of many native bacterial gene(s) (and consequent skewing towards high A+T content) results in the generation of sequences mimicking or duplicating plant gene control sequences that are known to be highly A+T rich. The presence of some A+T-rich sequences within the DNA of gene(s) introduced into plants (e.g., TATA box regions normally found in gene promoters) may result in aberrant transcription of the gene(s). On the other hand, the presence of other regulatory sequences residing in the transcribed mRNA (e.g., polyadenylation signal sequences (AAUAAA), or sequences complementary to small nuclear RNAs involved in pre-mRNA splicing) may lead to RNA instability. Therefore, one goal in the design of genes encoding palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans for maize expression, more preferably referred to as plant optimized gene(s), is to generate a DNA sequence having a higher G+C content, and preferably one close to that of maize genes coding for metabolic enzymes. Another goal in the design of the plant optimized gene(s) encoding palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans is to generate a DNA sequence in which the sequence modifications do not hinder translation.
The table below (Table 1) illustrates how high the G+C content is in maize. For the data in Table 1, coding regions of the genes were extracted from GenBank (Release 71) entries, and base compositions were calculated using the MacVector(trademark) program (IBI, New Haven, Conn.). Intron sequences were ignored in the calculations.
Due to the plasticity afforded by the redundancy of the genetic code (i.e., some amino acids are specified by more than one codon), evolution of the genomes in different organisms or classes of organisms has resulted in differential usage of redundant codons. This xe2x80x9ccodon biasxe2x80x9d is reflected in the mean base composition of protein coding regions. For example, organisms with relatively low G+C contents utilize codons having A or T in the third position of redundant codons, whereas those having higher G+C contents utilize codons having
G or C in the third position. It is thought that the presence of xe2x80x9cminorxe2x80x9d codons within a mRNA may reduce the absolute translation rate of that mRNA, especially when the relative abundance of the charged tRNA corresponding to the minor codon is low. An extension of this is that the diminution of translation rate by individual minor codons would be at least additive for multiple minor codons. Therefore, mRNAs having high relative contents of minor codons would have correspondingly low translation rates. This rate would be reflected by subsequent low levels of the encoded protein.
In reengineering genes encoding palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans for maize expression, the codon bias of the plant has been determined. The codon bias for maize is the statistical codon distribution that the plant uses for coding its proteins and the preferred codon usage is shown in Table 2. After determining the bias, the percent frequency of the codons in the gene(s) of interest is determined. The primary codons preferred by the plant should be determined as well as the second and third choice of preferred codons. Afterwards, the amino acid sequence of palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans is reverse translated so that the resulting nucleic acid sequence codes for exactly the same protein as the native gene wanting to be heterologously expressed. The new DNA sequence is designed using codon bias information so that it corresponds to the most preferred codons of the desired plant. The new sequence is then analyzed for restriction enzyme sites that might have been created by the modification. The identified sites are further modified by replacing the codons with second or third choice with preferred codons. Other sites in the sequence which could is affect transcription or translation of the gene of interest are the exon:intron 5xe2x80x2 or 3xe2x80x2 junctions, poly A addition signals, or RNA polymerase termination signals. The sequence is further analyzed and modified to reduce the frequency of TA or GC doublets. In addition to the doublets, G or C sequence blocks that have more than about four residues that are the same can affect transcription of the sequence. Therefore, these blocks are also modified by replacing the codons of first or second choice, etc. with the next preferred codon of choice.
It is preferred that the plant optimized gene(s) encoding palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans contain about 63% of first choice codons, between about 22% to about 37% second choice codons, and between about 15% to about 0% third choice codons, wherein the total percentage is 100%. Most preferred the plant optimized gene(s) contains about 63% of first choice codons, at least about 22% second choice codons, about 7.5% third choice codons, and about 7.5% fourth choice codons, wherein the total percentage is 100%. The preferred codon usage for engineering genes for maize expression are shown in Table 2. The method described above enables one skilled in the art to modify gene(s) that are foreign to a particular plant so that the genes are optimally expressed in plants. The method is further illustrated in pending PCT application WO 97/13402, which is incorporated herein by reference.
In order to design plant optimized genes encoding palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans, the amino acid sequence of said protein is reverse translated into a DNA sequence utilizing a non-redundant genetic code established from a codon bias table compiled for the gene sequences for the particular plant, as shown in Table 2.
The resulting DNA sequence, which is completely homogeneous in codon usage, is further modified to establish a DNA sequence that, besides having a higher degree of codon diversity, also contains strategically placed restriction enzyme recognition sites, desirable base composition, and a lack of sequences that might interfere with transcription of the gene, or translation of the product mRNA. Said sequence produced using the methods described herein is disclosed as SEQ ID NO:12.
In another aspect of the invention, genes encoding the palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans are expressed from transcriptional units inserted into the plant genome. Preferably, said transcriptional units are recombinant vectors capable of stable integration into the plant genome and selection of transformed plant lines expressing mRNA encoding for said desaturase proteins are expressed either by constitutive or inducible promoters in the plant cell. Once expressed, the mRNA is translated into proteins, thereby incorporating amino acids of interest into protein. The genes encoding palmitate-CoA xcex94-9 desaturase from Aspergillus nidulans expressed in the plant cells can be under the control of a constitutive promoter, a tissue-specific promoter or an inducible promoter as described herein.
Several techniques exist for introducing foreign recombinant vectors into plant cells, and for obtaining plants that stably maintain and express the introduced gene. Such techniques include acceleration of genetic material coated onto microparticles directly into cells (U.S. Pat. No. 4,945,050 to Cornell and U.S. Pat. No. 5,141,131 to DowElanco, now Dow AgroSciences). In addition, plants may be transformed using Agrobacterium technology, see U.S. Pat. No. 5,177,010 to University of Toledo, U.S. Pat. No. 5,104,310 to Texas AandM, European Patent Application 0131624B1, European Patent Applications 120516, 159418B1 and 176,112 to Schilperoot, U.S. Pat. Nos. 5,149,645, 5,469,976, 5,464,763 and 4,940,838 and 4,693,976 to Schilperoot, European Patent Applications 116718, 290799, 320500 all to Max is Planck, European Patent Applications 604662,627752 and U.S. Pat. No. 5,591,616 to Japan Tobacco, European Patent Applications 0267159, and 0292435 and U.S. Pat. No. 5,231,019 all to Ciba Geigy, now Novartis, U.S. Pat. Nos. 5,463,174 and 4,762,785 both to Calgene, and U.S. Pat. Nos. 20 5,004,863 and 5,159,135 both to Agracetus. Other transformation technology includes whiskers technology, see U.S. Pat. Nos. 5,302,523 and 5,464,765 both to Zeneca. Electroporation technology has also been used to transform plants, see WO 87/06614 to Boyce Thompson Institute, U.S. Pat. Nos. 5,472,869 and 5,384,253 both to Dekalb, W09209696 and W09321335 both to Plant Genetic Systems. Furthermore, viral vectors can also be used in produce transgenic plants expressing the protein of interest. For example, monocotyledonous plant can be transformed with a viral vector using the methods described in U.S. Pat. No. 5,569,597 to Mycogen and Ciba-Giegy, now Novartis, as well as U.S. Pat. Nos. 5,589,367 and 5,316,931, both to Biosource. All of these transformation patents and publications are incorporated herein by reference.
As mentioned previously, the manner in which the DNA construct is introduced into the plant host is not critical to this invention. Any method which provides for efficient transformation may be employed. For example, various methods for plant cell transformation are described herein and include the use of Ti or Ri-plasmids and the like to perform Agrobacterium mediated transformation. In many instances, it will be desirable to have the construct used for transformation bordered on one or both sides by T-DNA borders, more specifically the right border. This is particularly useful when the construct uses Agrobacterium tumefaciens or Agrobacterium rhizogenes as a mode for transformation, although T-DNA borders may find use with other modes of transformation.
Where Agrobacterium is used for plant cell transformation, a vector may be used which may be introduced into the host for homologous recombination with T-DNA or the Ti or Ri plasmid present in the host. Introduction of the vector may be performed via electroporation, tri-parental mating and other techniques for transforming gram-negative bacteria which are known to those skilled in the art. The manner of vector transformation into the Agrobacterium host is not critical to this invention. The Ti or Ri plasmid containing the T-DNA for recombination may be capable or incapable of causing gall formation, and is not critical to said invention so long as the vir genes are present in said host.
In some cases where Agrobacterium is used for transformation, the expression construct being within the T-DNA borders will be inserted into the plasmid pDAB1542 as described herein or into a broad spectrum vector such as pRK2 or derivatives thereof as described in Ditta et al., (PNAS USA (1980) 77:7347-7351 and EPO 0 120 515, which are incorporated herein by reference. Included within the expression construct and the T-DNA will be one or more markers as described herein which allow for selection of transformed Agrobacterium and transformed plant cells. The particular marker employed is not essential to this invention, with the preferred marker depending on the host and construction used.
For transformation of plant cells using Agrobacterium, explants may be combined and incubated with the transformed Agrobacterium for sufficient time to allow transformation thereof. After transformation, the agrobacteria are killed by selection with the appropriate antibiotic and plant cells are cultured with the appropriate selective medium. Once calli are formed, shoot formation can be encourage by employing the appropriate plant hormones according to methods well known in the art of plant tissue culturing and plant regeneration. However, a callus intermediate stage is not always necessary. After shoot formation, said plant cells can be transferred to medium which encourages root formation thereby completing plant regeneration. The plants may then be grown to seed and said seed can be used to establish future generations as well as provide a source for oil isolation. Regardless of transformation technique, the gene encoding palmitoyl-CoA xcex94-9 desaturase from Aspergillus nidulans is preferably incorporated into a gene transfer vector adapted to express said gene in a plant cell by including in the vector a plant promoter regulatory element, as well as 3xe2x80x2 non-translated transcriptional termination regions such as Nos and the like.
In addition to numerous technologies for transforming plants, the type of tissue which is contacted with the foreign genes may vary as well. Such tissue would include but would not be limited to embryogenic tissue, callus tissue types I, II, and III, hypocotyl, meristem, and the like. Almost all plant tissues may be transformed during dedifferentiation using appropriate techniques described herein.
Another variable is the choice of a selectable marker. Preference for a particular marker is at the discretion of the artisan, but any of the following selectable markers may be used along with any other gene not listed herein which could function as a selectable marker. Such selectable markers include but are not limited to aminoglycoside phosphotransferase gene of transposon Tn5 (Aph II) which encodes resistance to the antibiotics kanamycin, neomycin and G418, as well as those genes which encode for resistance or tolerance to glyphosate; hygromycin; methotrexate; phosphinothricin (bialophos); imidazolinones, sulfonylureas and triazolopyrimidine herbicides, such as chlorsulfuron; bromoxynil, dalapon and the like.
In addition to a selectable marker, it may be desirous to use a reporter gene. In some instances a reporter gene may be used with or without a selectable marker. Reporter genes are genes which are typically not present in the recipient organism or tissue and typically encode for proteins resulting in some phenotypic change or enzymatic property. Examples of such genes are provided in K. Weising et al. Ann. Rev. Genetics, 22, 421 (1988), which is incorporated herein by reference. Preferred reporter genes include the beta-glucuronidase (GUS) of the uida locus of E. coli, the chloramphenicol acetyl transferase gene from Tn9 of E. coli, the green fluorescent protein from the bioluminescent jellyfish Aequorea victoria, and the luciferase genes from firefly Photinus pyralis. An assay for detecting reporter gene expression may then be performed at a suitable time after said gene has been introduced into recipient cells. A preferred such assay entails the use of the gene encoding beta-glucuronidase (GUS) of the uida locus of E. coli as described by Jefferson et al., (1987 Biochem. Soc. Trans. 15, 17-19) to identify transformed cells.
In addition to plant promoter regulatory elements, promoter regulatory elements from a variety of sources can be used efficiently in plant cells to express foreign genes. For example, promoter regulatory elements of bacterial origin, such as the octopine synthase promoter, the nopaline synthase promoter, the mannopine synthase promoter; promoters of viral origin, such as the cauliflower mosaic virus (35S and 19S), 35T (which is a re-engineered 35S promoter, see PCT/US96/1682; WO 97/13402 published Apr. 17, 1997) and the like may be used. Plant promoter regulatory elements include but are not limited to ribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu), beta-conglycinin promoter, beta-phaseolin promoter, ADH promoter, heat-shock promoters and tissue specific promoters.
Other elements such as matrix attachment regions, scaffold attachment regions, introns, enhancers, polyadenylation sequences and the like may be present and thus may improve the transcription efficiency or DNA integration. Such elements may or may not be necessary for DNA function, although they can provide better expression or functioning of the DNA by affecting transcription, mRNA stability, and the like. Such elements may be included in the DNA as desired to obtain optimal performance of the transformed DNA in the plant. Typical elements include but are not limited to Adh-intron 1, Adh-intron 6, the alfalfa mosaic virus coat protein leader sequence, the maize streak virus coat protein leader sequence, as well as others available to a skilled artisan.
Constitutive promoter regulatory elements may also be used thereby directing continuous gene expression in all cells types and at all times (e.g., actin, ubiquitin, CaMV 35S, and the like). Tissue specific promoter regulatory elements are responsible for gene expression in specific cell or tissue types, such as the leaves or seeds (e.g., zein, oleosin, napin, ACP, globulin and the like) and these may also be used.
Promoter regulatory elements may also be active during a certain stage of the plants"" development as well as active in plant tissues and organs. Examples of such include but are not limited to pollen-specific, embryo specific, corn silk specific, cotton fiber specific, root specific, seed endosperm specific promoter regulatory elements and the like. Under certain circumstances it may be desirable to use an inducible promoter regulatory element, which is responsible for expression of genes in response to a specific signal, such as: physical stimulus (heat shock genes); light (RUBP carboxylase); hormone (Em); metabolites; chemical; and stress. Other desirable transcription and translation elements that function in plants may be used. Numerous plant-specific gene transfer vectors are known in the art.
One of the issues regarding exploiting transgenic plants having altered saturate levels is the expression of multiple chimeric genes at once. European Patent Application 0400246A1 describes transformation of two Bt genes in a plant; however, these could be any two genes or fragments thereof in either the sense or antisense orientation. For example, commercially available hybrids have now been produced having stacked traits such as herbicide and insect resistance. The options could include but are not limited to genes and fragments encoding the palmitoyl-CoA xcex94-9 desaturase from Aspergillus nidulans with acyl-ACP thioesterase genes or genes encoding proteins such as stearoyl-ACP desaturase, xcex2-ketoacyl synthase II and the like, as well as genes to impart insect control or herbicide resistance. Another way to produce a transgenic plant having multiple traits is to produce two plants, with each plant containing the oil modifying gene of interest. These plants can then be back-crossed using traditional plant breeding techniques available and well-known to those skilled in the art to produce plants wherein phenotypic characteristics are related to the presence of more than one chimeric gene.
The particular embodiments of this invention are further exemplified in the Examples. However, those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention as described more fully in the claims which follow thereafter.